This invention relates to diagnostic and therapeutic methods based upon the development of cellular immunity to immune privileged antigens and its role in the etiology of paraneoplastic neuronal disorders and tumor immunity, among other conditions.
Constant surveillance of epitopes throughout those structures in the body accessible to the immune system provides a very effective means for recognizing and maintaining xe2x80x9cselfxe2x80x9d and destroying epitopes and their carriers which invade the body or arise pathologically, such as infectious microorganisms. One important role of immune surveillance is the recognition and destruction of neoplastic cells that are believed to arise continuously in the body and for the most part are eliminated by the immune system before becoming detectable. However, examples of naturally-occurring tumor immunity have been elusive. Cytotoxic T lymphocytes, key participants in effective immune surveillance, are not expanded in patients with active tumors, even when these tumors express what are believed to be tumor-specific antigens such as the MAGE/MART antigens of melanoma.
Effective tumor immunity has been documented, however, in individuals with paraneoplastic neuronal disorders (PNDs). These syndromes are poorly understood diseases in which serious effects of cancer in the body occur in the nervous system without any direct involvement of the tumor. PND patients typically present to physicians with neurologic dysfunction unaware that they harbor a tumor. For example, patients with ovarian or breast cancer who develop paraneoplastic cerebellar degeneration (PCD) have an effective tumor immune response (3,4,5; reviewed in 1,2,6), and moreover, the tumor expresses neuron-specific proteins (antigens). These patients have in circulation and in the cerebrospinal fluid (CSF) antibodies against these tumor cell antigens, which also cross-react with the same proteins expressed in neurons, termed onconeural antigens. A high titer antibody recognizes the intracellular antigen cdr2 expressed in the ovarian or breast tumor present in PCD patients (10); and also recognizes the antigen in Purkinje neurons of the cerebellum (10). However, as will be elaborated below, the existence of this antibody does not account for the etiology of the PND nor for effective tumor killing.
Certain regions of the body, such as the brain, eye, and testis, are protected from immune surveillance, these sites are referred to as immune privileged. Based on the above observations, the immune system is proposed to initiate PCD by recognizing the normally immune-privileged antigen cdr2 (10) when it is ectopically expressed in gynecologic tumors. This immune response is associated clinically with effective tumor immunity, and is believed to lead to the recognition and destruction of Purkinje neurons expressing cdr2. cDNAs encoding several of the target antigens have been cloned, for example, cdr2 which has been shown to be the correct tumor antigen (9,54). However, because the target neuronal antigen is cytoplasmic, the role of circulating and cerebrospinal fluid (CSF) antibodies against these antigens in the pathogenesis of PCD is questionable. Moreover, attempts to reproduce the disorder by passive or active transfer of antibodies have failed (11,12,13). As the target organ, the brain, is immune privileged, and furthermore the target antigen is cytoplasmic, the etiology of the paraneoplastic syndrome is difficult to reconcile. This is further confounded by the apparent absence of a cellular immune response against tumor antigens in general and the apparent absence of a cellular immune response in PCD. No cytotoxic T lymphocytes were found against the cdr2 protein using autologous dendritic cells in a patient with PCD (47). The etiology of tumor immunity in PND is enigmatic.
As described above, the paraneoplastic syndromes are serious conditions associated with tumors and frequently affect the central nervous system; these disorders are collectively referred to as paraneoplastic neuronal disorders (PND). For example, one common paraneoplastic disorder which is seen in patients with breast or ovarian cancer is paraneoplastic cerebellar degeneration, or PCD, in which a progressive and severe neurological dysfunction occurs involving the cerebellum, leading to dyscoordination of the legs and arms, dizziness and double vision. Frequently, these symptoms appear before the diagnosis of cancer. In another example of neurological degeneration, Hu syndrome is associated with small cell lung cancer and antibodies to the onconeural antigen Hu. In other examples, opsoclonus, or spontaneous, chaotic eye movements, and myoclonus, jerky body movements, may accompany breast cancer, fallopian tube cancer, or small cell lung cancer, and are associated with antibodies to the onconeural antigen Nova.
The target onconeural antigens have yet to be identified for some disorders believed to be paraneoplastic. Patients with Hodgkin""s disease and other lymphomas may develop subacute cerebellar degeneration that is believed to be immune mediated (22,42). Eaton-Lambert syndrome, a condition causing weakness in the limbs, may also accompany intrathoracic tumors such as lung cancer and is believed to be immune mediated (2). Some patients who develop spinal cord dysfunction (e.g., myelopathy), motor neuron diseases, blindness and other neurologic symptoms are found to have specific sets of underlying tumors and are believed to have immunity to unknown or partially-characterized onconeural antigens (2,37). Less well understood, the incidence of the muscle diseases dermatomyositis and polymyositis is increased in cancer patients. The dermatologic condition vitiligo, in which melanocytes producing skin pigment are destroyed, appears associated with a decrease in incidence of melanoma. It is thus apparent that an association exists between tumors, and in some cases tumor immunity, and the sites of the paraneoplastic disorder symptoms, perhaps through the existence of some common antigens.
Several lines of evidence suggest the existence of naturally-occurring tumor immunity in PND patients. PND-associated tumors are typically occult (24,25); in several cases they have been identified only by microscopic analysis of suspect organs following exploratory surgery or at autopsy. Patients with PND-associated tumors have significantly-limited disease and an improved tumor prognosis relative to patients with histologically-identical tumors unassociated with PND (20,24,26-28). In some cases PND-associated tumors have been documented to regress with the onset of autoimmune neurologic disease (7).
Specific clinical data regarding anti-tumor immunity is available for several of the PNDs. Patients with paraneoplastic encephalomyelitis harbor high titers of an antibody termed Hu and small cell lung cancers (SCLCa); their tumors are typically limited to single nodules (53/55 [96%] patients in the most complete study published [3]). This is a remarkable finding given that most SCLCa patients from unselected series (over 60%) have widely metastatic disease at the time of diagnosis (and no detectable titers of Hu antibody). In addition, fifteen percent of SCLCa patients without PND nonetheless have detectable titers of the Hu antibody (20). These patients have statistically significant increases in the frequency of limited stage disease, complete response to chemotherapy and longer survival (3,5). These results suggest that anti-PND antibodies may be associated with suppression of tumor growth independently from their association with neurologic disease.
There are also firm associations between the presence of the Nova (Ri) (28) and Yo (10) antibodies in PND patients and clinically-limited malignancy. Both antibodies are found in women with gynecologic cancer. Of 52 Yo-antibody-positive patients with breast or ovarian cancer (4), two-thirds (34/52) presented with neurologic symptoms prior to the diagnosis of cancer, and 87% (45/52) had limited oncologic disease when diagnosed; similarly, 4/7 Nova-positive patients presented with neurologic symptoms, 6/7 had limited stage disease, and no tumor could be found in one patient (28). By comparison, only 50-60% of unselected breast cancer patients, and 25% of ovarian cancer present with limited stage disease (8).
Experimental observations support the clinical evidence that there is immunologic recognition of tumor cells in PND. High titer anti-PND antibodies are found in the serum and cerebrospinal fluid of PND patients. In vitro, these antibodies react specifically with tumor specimens obtained from PND patients cells, as well as neurons from clinically affected areas of the nervous system (24,25, 29). For example, 10/10 breast or ovarian tumors from Yo-positive patients were immunoreactive with biotinylated Yo antisera (4), and 3/4 breast or fallopian tumors from Nova-positive patients were immunoreactive with biotinylated Nova antisera (28). Taken together, these observations suggest that PND antibodies are more than markers for neurologic disease or even the presence of tumor cells, but are markers, and perhaps in part reflective of effective anti-tumor immune responses.
The immunologic basis of the anti-tumor and antineuronal immune response in PND is unknown. The finding of autoantibodies with neuronal binding specificity, and observations on autoimmune neurologic disorders of the peripheral nervous system, have focused attention on the role of B cells in the pathogenesis of PND. In myasthenia gravis (MG) and Lambert-Eaton myasthenic syndrome (LEMS), antineuronal antibodies have been found to passively transfer autoimmune disease in animals (30, 31). In PND, there are relatively higher titers of antibody in the CSF than serum (IgG index greater than 1) (32) suggestive of an active B cell inflammatory response within the CNS compartment. Furthermore, although the data is not fully compelling, there have been numerous reports that PND antibodies may be neurotoxic in vitro and that antibodies may be able to be taken up by neurons (33,34). These observations have led clinicians to focus therapy for the PNDs on the elimination of PND antibodies. Unfortunately these attempts have been uniformly unsuccessful (24, 32, 35). Several features of the PNDs distinguish them from MG and LEMS and suggest that B cells might not be sufficient or even necessary for the development of PND. PND antigens have been found to be cytoplasmic (Yo, xcex2-NAP) or nuclear (Nova, Hu) proteins, unlike the target antigens in MG (the acetylcholine receptor) or LEMS (the presynaptic calcium channel) (2). It is difficult to reconcile these observations with the premise that PND antibodies play a primary role in PND autoimmunity. Moreover, attempts to produce animal models of PND, including infusion of antibody into the CSF and immunization with cloned fusion protein, have failed (11, 12).
Thus, the etiology of the paraneoplastic syndromes appears to have an immunological basis, heretofore undefined. It is towards a better understanding of the etiology of the paraneoplastic neuronal disorders and the establishment of a link between effective tumor immunity and these serious, remote complications of neoplasia in immune privileged sites that the present invention is directed, with objectives of improving the detection of tumors and paraneoplastic disorders in individuals in general and offering improved therapies for both tumors expressing immune privileged antigens and the associated syndromes.
The inventors herein have made the surprising and remarkable finding of the presence of tumor antigen-specific T lymphocytes (CTLs) in patients with paraneoplastic neuronal disorders. This finding provides a basis for understanding the desirable and often effective cell-based immunologic attack on the tumors, and the effective but undesirable attack on remote target organ(s) of the paraneoplastic disorders by CTLs. Expression of the same immune-privileged antigen by these remote tissues as that which is expressed in the tumor cells, and to which T lymphocytes are targeted, explains for the first time the etiology of the PNDs. Both activated CTLs and memory T lymphocytes specific for the tumor and for the remote antigen have been detected. This finding provides an appreciation that immune privileged antigens offer a unique set of targets for the immune system. If expressed in tumors, they provide targets for effective anti-tumor immunity. If immune-privilege or tolerance to these antigens is broken, for example in the setting of effective anti-tumor immunity, autoimmune disease may result. The identification of a cellular immune response to immune-privileged antigens that can be readily and specifically detected, amplified, or inhibited, provides the basis for diagnostic and therapeutic utilities disclosed herein. Based upon this discovery, diagnostic utilities are disclosed for the detection and monitoring of cellular immunity to privileged antigens, and therapeutic methods are described for increasing the effectiveness of anti-tumor immunity and also for protecting the immune privileged site from immune-mediated pathology. Known diagnostic and therapeutic procedures and manipulations of the immune system are modified based on the discoveries herein in order to detect and modulate the immune response to immune-privileged antigens.
As will be described in more detail, below, only a fraction of patients with a specific T lymphocyte response to immune privileged antigens, especially those with tumors, exhibit an overt paraneoplastic disorder, yet such patients are at risk for the development of, or may have as-yet undetected autoimmune disease or another subclinical disorders. In accordance with the present invention, methods for determining in an individual the presence and extent of a cellular immune response to an immune-privileged antigen are provided, the cellular immune response associated directly or indirectly with a pathological state. Examples of pathological states include but are not limited to dysproliferative diseases, paraneoplastic syndromes, and autoimmune disorders. The method comprises quantitating in a sample of bodily fluid from an individual the presence and extent of T lymphocytes specific for the immune-privileged antigen or its fragments. The preferred method involves the detection of T lymphocytes which recognize paraneoplastic antigens, and most preferably, onconeural antigens such as cdr2 and Hu antigen. One example of a means for detection comprises determining the extent of activation of T lymphocytes upon exposure to the antigen by measuring cytokine production; another method comprises detecting the extent of recognition by the cytotoxic T cells of target cells expressing the antigen. Methods for detecting T lymphocytes bearing receptors for immune-privileged antigen are also provided.
In the instance where the T lymphocytes to be detected are memory T cells, the methods comprises detecting the extent of activation of memory T cells after exposure to antigen-presenting cells (APCs) presenting the immune-privileged antigen. In another embodiment, the extent of recognition of target cells expressing the antigen is determined after exposure of the memory T lymphocytes to APCs presenting the immune-privileged antigen.
The present invention further provides a method for screening individuals for the presence of tumors expressing immune-privileged antigens as well as detecting the early onset or propensity to develop a pathological state caused by a cellular immune response to an immune-privileged antigen. This method comprises measuring the presence and extent of T lymphocytes specific for immune privileged antigens. Furthermore, a method is provided for determining whether a neoplasm expresses an immune-privileged antigen by quantitating T lymphocytes that are specific for the antigen or its fragment. In another embodiment, a method is disclosed for determining whether a patient with a immune-privileged antigen-expressing tumor has a sufficient population of antigen-specific T lymphocytes to control the tumor or is a candidate for anti-cancer therapy. This method comprises quantitating T lymphocytes specific for the antigen or a fragment. In a still further embodiment, a method for monitoring the effectiveness of therapies directed to modulate the population of immune-privileged antigen-specific T lymphocytes in a patient is described wherein the numbers of antigen-specific T lymphocytes are quantitated.
The cDNAs encoding the target immune-privileged as well as their expressed proteins and fragments thereof may be used in the present invention to provide reagents for carrying out the diagnostic and therapeutic methods as described herein, as well as being part of a diagnostic kit. As described above, the sequence and cDNA of cdr2 is known (9,54); its fragments that complexes with HLA are described below.
In a further example of a screening method for identifying the number of immune-privileged antigen-specific T cells in a patient sample, the following steps may be carried out:
i) maturing dendritic cells in the blood sample;
ii) exposing the matured dendritic cells to apoptotic debris from unrelated cells expressing an immune-privileged antigen;
iii) co-incubating the immune-privileged antigen-exposed dendritic cells with the peripheral blood lymphocytes from the patient; and
iv) correlating the amount of interferon-xcex3 released from the lymphocytes with the number of immune privileged antigen-specific T cells in the sample.
By way of non-limiting example, the immune-privileged antigen may be cdr2. The unrelated cells expressing an immune-privileged antigen may be cells stably transfected to express an immune-privileged antigen, such as cdr2. The interferon-xcex3 release may be measured in an ELISPOT assay.
Diagnostic kits are also provided with componentry capable of measuring the above-described T lymphocytes and antigens comprising, for example, one or more of the following reagents: an isolated, immune-privileged antigen or preferably a fragment of the immune-privileged antigen; a target cell expressing the immune-privileged antigen or its fragment; a fragment of the immune-privileged antigen in a tetrameric complex with HLA; and a reagent such as an antibody or labeled antibody which recognizes a fragment of the immune-privileged antigen in a complex with HLA. When the immune-privileged antigen is cdr2, useful isolated polypeptide sequences identified include cdr2 peptides referred to as Yo1 through Yo8, or cdr2-1 through cdr2-8, and identified herein as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. The kit may include target cells prepared from a cell line or, for example, Drosophila, which expressed the immune-privileged antigen, and further may express HLA molecules and co-stimulatory molecules. A kit may further include components for detecting cytokine production, such as xcex3-IFN, as a means for detecting immune cell activation. To broadly screen samples from a variety of patients with different HLA haplotypes, a variety of target cells expressing the same immune-privileged antigen, but different HLA haplotypes, may be employed in order to detect immune-privileged antigen specific T lymphocytes regardless of the patient""s HLA haplotype.
It is another object of the present invention to provide methods for treating a neoplasm in a patient in which the neoplasm expresses an immune-privileged antigen. One preferred embodiment is accomplished by increasing the number of immune-privileged, antigen-specific cytotoxic T lymphocytes present in the patient. In one, non-limiting example, the method is carried out by first isolating a quantity of APCs from a sample of the patient""s blood, then exposing the APCs in vitro to the immune-privileged antigen or its fragment, followed by reintroducing the antigen-exposed APCs to the patient. In another related embodiment, the same method is followed with an additional step of exposing the antigen-exposed APCs in vitro to a quantity of T lymphocytes isolated from the patient, and reintroducing the T lymphocytes to the patient. These examples are illustrative of methods of providing the patient with immune privileged antigen-specific T lymphocytes and/or immune-privileged antigen-presenting APCs in order to develop or enhance immunity to the tumor.
Methods for achieving presentation of the immune-privileged antigen or its fragment on the APCs in the aforementioned methods is achieved using any one of several methods. For example, APCs are provided with apoptotic cells expressing the immune-privileged antigen or a fragment. These can be commonly available cell lines expressing the immune privileged antigen, such as HeLa cells, which express the cdr2 antigen (9), or transfected cells such as Drosophila cells expressing the gene encoding the immune-privileged antigen. These cells may also be further engineered to additionally express the gene encoding the MHC molecule haplotype of the patient, and even further engineered to express co-stimulatory molecules, such that the Drosophila cells function as an antigen-presenting cell, thus forming a useful APC for in-vivo or ex-vivo stimulation of T lymphocytes as described above. These cells also have diagnostic utility, as described herein. The preferred antigen is a paraneoplastic antigen, and most preferred, an onconeural antigen such as cdr2 and Hu antigen. In a further embodiment, the immune-privileged antigen-specific T lymphocytes are derived from a donor individual of the same HLA haplotype as the patient.
In a further embodiment of the present invention, a method for treating a pathological state in a mammal is provided, wherein the pathological state is caused by the presence in the mammal of T lymphocytes specific for an immune-privileged antigen. The method consists of administration of an effective amount of an agent which decreases the population of activated T lymphocytes specific for cells expressing the immune-privileged antigen. Non-limiting examples of such agents include tacrolimus, cyclosporin, immunosuppressive cytokines, corticosteroids, and combinations. The preferred agent is tacrolimus. The immune-privileged antigen is preferably a paraneoplastic antigen, most preferably, and onconeural antigen such as cdr2 and Hu antigen and their fragments. The preferred route of administration of the agents is to the central nervous system. Other effective routes of administration are also disclosed.
In a further embodiment of the present invention, a method is provided for decreasing the ability of non-tumor cells expressing privileged antigens to be killed by cytotoxic T lymphocytes as well as decreasing the expression of paraneoplastic antigens on non-tumor cells. These may be achieved by several methods, for example, by reducing the cytokine level in contact with the affected cells; increasing the expression of Nef or Nef-like proteins, inhibiting perforin-mediated CTL killing of neurons, and inhibiting apoptosis of the target cells.
In another embodiment, methods and agents are provided for enhancing the killing of tumors expressing immune privileged antigens by T lymphocytes. These methods include administering cytokines, inhibiting Fas-ligand expression in the tumor, and inducing the expression of MHC I molecules on the tumor. Other methods may be used in combination with increasing the immune-privileged T lymphocyte activity in the patient.
In a preferred embodiment, an individual with a tumor expressing an immune-privileged antigen and also suffering from a paraneoplastic disease or other syndrome in which the immune system is recognizing and attacking the same antigen at a non-tumor site within the body is treated by increasing the immune recognition of the immune-privileged antigen of the tumor exemplified by the non-limiting examples of methods disclosed herein, while concurrently protecting the non-tumor site from immune attack by the corresponding methods disclosed herein.
It is thus a principal object of the present invention to take advantage of the presence of immune-privileged antigen-specific T lymphocytes to detect the existence of a pathological state in a patient and to monitor the efficacy of treatments based upon the enhancement of tumor immunity by T lymphocytes as well as their suppression in the treatment of the associated syndrome in the non-tumor site. It is a further object of the present invention to provide both diagnostic and therapeutic purposes for the detection of tumors and paraneoplastic syndromes, to increase the immune destruction of such tumors as well as to protect the non-tumor organs susceptible to disease caused by the same T lymphocytes.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.